Paxillin is a cytoskeletal phosphoprotein involved in actin-membrane attachment at sites of cell adhesion in muscle and non-muscle cells. Our cloning of the paxillin cDNA indicates that this protein contains a series of distinct domains implicated in protein-protein interactions. This proposal is designed to determine which regions of the molecule are involved in targeting paxillin to specialized sites of cell attachment to the extracellular matrix known as focal adhesions. Proteins interacting with the individual domains of paxillin will be identified and characterized. The role of paxillin phosphorylation in regulating these associations will also be studied. Specifically, the region(s) of paxillin necessary for targeting the protein to focal adhesions will be determined through stable transfection of defined regions of avian paxillin cDNA into NIH 3T3 or CHO cells. Expression and subcellular localization of the avian paxillin will be monitored by immunoprecipitation and immunofluorescence microscopy respectively, using chicken-specific paxillin antisera. Alternatively, segments of paxillin cDNA will be generated by polymerase chain reaction and the corresponding protein fragments expressed in bacteria as glutathione S-transferase (GST) fusion proteins. The purified fusion proteins will be microinjected into mammalian cells and localized with anti-GST antibodies. Evidence for focal adhesion disruption, resulting from over-expression of particular paxillin constructs, will also be monitored by immunofluorescence microscopy. The same GST-paxillin fusion proteins will be used as affinity matrices in precipitation binding assays to identify and isolate novel paxillin binding proteins using fibroblast and smooth muscle lysates. Binding proteins will be detected using a combination of metabolic labeling, blotting and in vitro kinase assays. Additionally, the yeast Interaction Trap system will be used to identify and clone directly, paxillin binding proteins. Any novel proteins detected will be further purified and characterized biochemically. Particular attention will be directed towards further characterization of three paxillin binding proteins; an unidentified 100 kDa phosphoprotein, a serine kinase and a threonine kinase. Paxillin phosphorylation resulting from the activity in vitro of the paxillin-associated kinases will be examined by deletion and site-directed mutagenesis of the paxillin cDNA followed by phosphopeptide and phosphoamino acid analysis to identify target amino acids. These data will be correlated with a similar analysis of paxillin phosphorylation induced in vivo in fibroblasts stimulated by attachment/detachment to/from the extracellular matrix. The importance of the phosphorylated residues in focal adhesion organization and paxillin targeting will be addressed by transfection of paxillin cDNA containing point mutations of the appropriate amino acids. Data derived from these experiments are expected to contribute to our understanding of the mechanisms regulating cytoskeletal assembly associated with cell adhesion, thereby providing a foundation for determining the role of such events in mediating signals, derived from the extracellular environment, that lead to gene expression and cell proliferation.